The selective and quantitative detection of trace amounts of Zn(II) or Cd(II) is commercially desirable for the diagnosis of metal ion induced diseases and in protecting the environment.
Zinc is an essential element which is present in the body at approximately 1 micromole/L. The USDA recommended dietary intake of Zn(II) is only 15 mg/day, which indicates how little Zn(II) is required to maintain the required level of this element in a healthy adult. Despite this relatively low concentration, Zn(II) plays an essential role in biology and nutrition. Minor perturbations of normal Zn(II) levels have been associated with retarded sexual maturation, stunted growth, and skin damage. Over 99% of Zn(II) in biological tissues and fluids is present in a chemically combined form, with very little present as free Zn(II). Traditional methods such as atomic absorption effectively measure total Zn(II) but cannot distinguish between the chemically combined and the free forms. The problem of detecting free Zn(II) is compounded because total free Zn(II) is decreased only very slightly (50-100 pmol/106 cells) in cases of severe Zn(II) deficiency.
Zinc is the second most abundant transition metal in the brain. Zinc is essential for brain maturation and function. Approximately ninety percent of cellular zinc is bound to metalloproteins, while the remainder is localized at presynaptic vesicles in the ionic or loosely bound form. Vesicular zinc is thought to play an important role in synaptic neurotransmission. Several devastating cerebral disorders, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS) are associated with abnormally high vesicular Zn(II) concentration (Cuajungo et al., 1997). Because of its association with major neurological disorders, zinc imaging becomes an increasingly important tool in brain research. In particular, fluorescence microscopy is a very useful technique for monitoring real-time zinc distribution.
Cadmium, both as the free metal and in its compounds, is highly toxic, and has been designated one of the 100 most hazardous substances under Section 110 of the Superfund Amendments and Reauthorization Act of 1986. Poisoning occurs with by ingestion or by inhalation.
Chemical pneumonitis or pulmonary edema may result from acute exposure to cadmium fumes, as oxide or chloride aerosols, at a dose of 5 mg/m3 over an eight hour period. Acute ingestion of cadmium concentrations above about 15 ppm produce symptoms of nausea, vomiting, abdominal cramps, and headache. Possible sources of such poisoning have been traced to cadmium-plated cooking utensils, cadmium solders in water coolers, or from acid juices stored in ceramic pots glazed using cadmium-treated compounds.
Most biological molecules do not fluoresce on their own, so they must be linked with fluorescent molecules, or fluorochromes, in order to create specific fluorescent probes. The feasibility of using fluorescence technology for a particular application is often limited by the availability of an appropriate fluorescent sensor. There are a number of features that are desirable in fluorescent sensors, some of which may or may not be present in any particular sensor.
First, fluorescent sensors should produce a perceptible change in fluorescence upon binding a desired analyte. Second, fluorescent sensors should selectively bind a particular analyte. Third, to allow concentration change to be monitored, fluorescent sensors should have a Kd near the median concentration of the species under investigation. Fourth, fluorescent sensors, especially when used intracellularly, should produce a signal with a high quantum yield. Fifth, the wavelengths of both the light used to excite the fluorescent molecule (excitation wavelengths) and of the emitted light (emission wavelengths) are often important. If possible, for intracellular use, a fluorescent sensor should have excitation wavelengths exceeding 340 nm to permit use with glass microscope objectives and prevent UV-induced cell damage, and possess emission wavelengths approaching 500 nm to avoid autofluorescence from native substances in the cells and allow use with conventional fluorescence microscopy optical filter sets. Finally, ideal sensors should allow for passive and irreversible loading into cells.
Since the Zn(II) ion is spectroscopically silent, fluorescence microscopy for Zn(II) requires a sensor that makes it possible to observe this ion. There are several requirements that a fluorescent sensor for zinc needs to meet. First of all, it must produce a strong fluorescent signal upon binding the analyte. Secondly, the sensor needs to exhibit strong zinc binding, ideally having an apparent dissociation constant, Kd, near the median of Zn(II) concentration. The latter requirement is particularly challenging, given that Zn(II) concentration is known to be as low as femtomolar (Hitomi et al., 2001). Strong selectivity is another important factor in Zn(II) detection, because Zn(II) concentration is typically six to seven orders of magnitude lower than the concentration of the more abundant divalent metal ions such as Mg(II) and Ca(II) (Fraustro da Silva et al., 1993). Finally, there are several biological requirements to prevent cell damage from excitation and emission wavelengths, as noted above. In addition to that, the sensor must be soluble in physiological media.
The detection of Zn(II) or Cd(II) in the environment is also important, and is presently an intractable problem. For example, interest in Zn(II) concentrations in the ocean stems from its dual role as a required nanonutrient and as a potential toxic agent due to its widespread industrial and marine usage. Zinc exists at natural levels in ocean surface water at a total concentration of about 0.1 nM. Dissolved Zn(II) concentrations in seawater have been determined using atomic absorption spectrometry, mass spectrometry and voltammetry. The concentration data are inaccurate because of interference from other cations naturally present in sea water. A rapid, selective an more sensitive test for Zn(II) or Cd(II) is desirable.
A limited number of fluorescent sensors possess these desirable properties.